Alkyl benzidine compounds, including dialkyl benzidine compounds and tetraalkyl benzidine compounds such as 3,3′,5,5′-tetramethylbenzidine (TMB) have many uses. For example, such compounds can be used as colorimetric reagents to measure the amount of free chlorine present in an aqueous solution, for example as described in U.S. Pat. No. 5,972,713, which is incorporated by reference herein in its entirety, or as colorimetric substrates for use in enzyme-linked immunosorbent assays (ELISA), in which the colorimetric substrate is, for example, oxidized by an enzyme such as horseradish peroxidase to result in the generation of a visible colour change.
Chlorine is frequently used as a disinfecting agent to control microorganisms that may be present in domestic water supplies, including drinking water and water used for swimming or bathing. A disinfecting level of chlorine would include a level of approximately 1 part per million (ppm) chlorine. As an example, a recommended chlorine concentration is about 1 ppm in swimming pools and is about 3 ppm in hot tubs.
Various sources of chlorine can be used for water disinfection, including chlorine gas (Cl2), calcium hypochlorite (Ca(OCl)2), or sodium hypochlorite (NaOCl). All of these sources form hypochlorous acid (HClO) in water. Hypochlorous acid is the principal effective disinfecting agent when chlorine is used as the disinfecting agent.
Reactions can occur that affect the performance of hypochlorous acid as a disinfecting agent. First, chlorine can react with hydroxide ion (OH−), which is readily available in aqueous solutions having an alkaline pH to form a hypochlorite ion (OCl−). A hypochlorite ion is only about two-thirds as effective as a disinfecting agent as compared with hypochlorous acid. The sum of hypochlorous acid and hypochlorite ions present in a solution is referred to as the free chlorine.
Second, chlorine can react with ammonia (NH3) and organic nitrogen compounds including proteins and amino acids to form chloramines, as monochloramine (NH2Cl), dichloramine (NHCl2) and nitrogen trichloride (NCl3). Chloramines are less effective as a disinfecting agent. The sum of the chloramine species present in a solution is referred to as the combined chlorine. Total chlorine is the sum of both the free chlorine and the combined chlorine present in a solution.
In order to ensure that a sufficient amount of chlorine is present in a water sample to permit the safe consumption or use of the water, testing is frequently conducted. Many existing tests to measure the amount of chlorine present in water use N,N-diethyl-p-phenylenediamine (DPD) or o-tolidine. However, concerns have arisen regarding the toxicity of these compounds, meaning that alternative assays are needed.
Because chlorine is toxic and excess of chlorine can cause health problems, chlorine concentration is often monitored and controlled. For example, the chlorine concentration in most disinfected drinking water is about 0.2-1 ppm, and the chlorine concentration in dialysis water is even lower and may be e.g. less than 0.1 ppm. If there is too much chlorine present in dialysis water, there can be extremely severe effects on the patient. As an example, in one instance a patient died when a chlorine test of dialysis water with diethyl-p-phenylene diamine (DPD) yielded a false negative result due to bleaching of the DPD.
3,3′,5,5′-tetramethlybenzidine (TMB) is an exemplary alkyl benzidine compound that is a colorimetric substrate that can be used in various assays, including chlorine testing, enzyme-linked immunosorbent assays (ELISA), and immunohistochemistry. TMB can act as a hydrogen donor for the reduction of hydrogen peroxide to water by peroxidases such as horseradish peroxidase. This causes the solution to change to a blue colour. The reaction can be stopped by the addition of acid or another stop reagent, which turns the TMB a yellow colour.
Many alkyl benzidine compounds, including dialkyl benzidine compounds and tetraalkyl benzidine compounds such as TMB, are not soluble in water. Traditional methods of dissolving alkyl benzidine compounds such as TMB have used solvents such as dimethyl sulfoxide (DMSO) or dimethylformamide (DMF). Even when dissolved in such solvents, solutions of TMB developed to date do not exhibit good storage stability. Thus, when TMB is to be used in an assay, the solution of the alkyl benzidine compound, e.g. TMB, to be used must be freshly prepared. This is inconvenient to those carrying out the assay.
A stable formulation of TMB has been previously disclosed by the inventor in U.S. Pat. No. 9,714,939, which is incorporated by reference herein in its entirety. That formulation still incorporates a significant proportion (10% v/v) of DMSO as a solvent, and has the TMB being initially dissolved into the DMSO solvent.
There is a general desire for improved formulations of alkyl benzidine compounds, including dialkyl benzidine compounds and tetraalkyl benzidine compounds.
The foregoing examples of the related art and limitations related thereto are intended to be illustrative and not exclusive. Other limitations of the related art will become apparent to those of skill in the art upon a reading of the specification and a study of the drawings.